It is not always practical or desirable to transfuse a patient with donated blood. In these situations, use of a red blood cell substitute is desirable. Such a product would need to transport oxygen, just as red blood cells do.
When patients lose blood, it is usually necessary to replace the entire fluid volume lost. However, it is not usually necessary to replace all of the lost hemoglobin. The primary goal of hemoglobin replacement therapy is to transport oxygen from the lungs to peripheral tissues. Hemoglobin administration also increases and maintains plasma volume and decreases blood viscosity. While many volume expanding colloid and crystalloid solutions are now marketed, none can transport oxygen. The only current therapy with this capability is human blood transfusion.
Genetic engineering techniques have allowed the expression of heterologous proteins in a number of biological expression systems, for example, insect cell lines, transgenic cells, yeast systems and bacterial systems. Expression of hemoglobin in particular has been demonstrated in transgenic pigs (Logan et al., WO 92/22646), yeast (De Angelo et al., WO 93/08831 and WO 91/16349; Hoffman et a., WO 90/13645), and the bacterial E. coli system (Hoffman et al., WO 90/13645). Although expression of hemoglobin in these heterologous systems can be achieved at useful levels, purification of the final product to the extreme level of purity required for practical use of hemoglobin remains difficult. Removal of contaminating isoforms of hemoglobin is particularly difficult in that these isoforms often co-purify with the desirable form of hemoglobin.
Hemoglobin (Hb) is a tetrameric protein molecule composed of two alpha and two beta globin units. Alpha and beta globin subunits associate to form two stable alpha/beta dimers, which in turn loosely associate to form the hemoglobin tetramer. Human hemoglobin Ao (also known as naturally occurring or native hemoglobin) is a heterotetramer composed of two alpha globin subunits (a.sub.1, a.sub.2) nd two beta globin subunits (b.sub.1, b.sub.2). There is no sequence difference between a.sub.1 and a.sub.2 or b.sub.1 and b.sub.2. In the unoxygenated ("deoxy", or "T" for "tense") state, the subunits form a tetrahedron. The a.sub.1 b.sub.1 and a.sub.2 b.sub.2 interfaces remain relatively fixed during oxygen binding, while there is considerable flux at the a.sub.1 b.sub.2 and a.sub.2 b.sub.1 interfaces. In the oxygenated ("oxy" or "R" or relaxed) state, the intersubunit distances are increased. The subunits are noncovalently associated by Van der Waals forces, hydrogen bonds and, for deoxy Hb, salt bridges.
In fully functional normal or native hemoglobin, a heme molecule is incorporated into each of the alpha and beta globins. Hence is a large organic molecule coordinated around an iron atom. Heme is also a cofactor in hemoglobin and is required to form soluble hemoglobin. A heme group that is lacking the iron atom is known as protoporphyrin IX (PIX) and is non-functional (cannot bind a ligand). PIX can be incorporated into one or more of the a and b subunits of hemoglobin, but the PIX-containing subunit lacks the ability to bind and release oxygen or other ligands. If all of the prosthetic groups are protoporphyrin IX rather than heme, then the hemoglobin cannot bind or release oxygen and therefore is functionless.
In E. coli and related bacteria, heme.sub.b (which is also known as "protoheme," "ferrous protoporphyrin IX" and also referred to herein as "heme") essential for respiration and for detoxification of reactive species of oxygen. Heme.sub.b serves as an essential cofactor for b-type cytochromes, catalase and peroxidase.
Biosynthesis of heme.sub.b occurs via a complex, branched pathway that involves up to twelve gene products. In non-recombinant E. coli cells, the accumulatin of large pools of heme pathway intermediates or free heme is deleterious to the cells. For example, E. coli cells with certain mutants in the gene encoding hemH are impaired in their ability to insert iron into PIX. Such hemH mutants accumulate large pools of PIX and are light sensitive. Miyamoto et al., J. Mol. Biol., 219:393-398 (1991) and Miyamoto et al., FEBS Letter, 310:246-248 (1992).
E. coli cells into which an extra copy of the hemH gene has been inserted show a decrease in the amount of PIX in the cell. Kanazireva, et al., J. of Bact., 177:6693-6694 (1995).
Because heme has a propensity to cause oxidative damage to the lipid and protein components of cellular membranes, heme is normally found associated with proteins in the cell, rather than as free heme.
The hemH locus represents the final gene in the heme biosynthesis pathway of E. coli. The gene codes for the ferrochelatase protein which is responsible for adding iron into the precursor porphyrin molecules thereby converting PIX to heme.
As discussed above, PIX incorporation produces reduced function or functionless hemoglobin. Therefore, a need exists for controlling PIX formation in recombinant hemoglobin. The present invention satisfies this need and provides related advantages as well.